Alcoholism and alcohol abuse is a major cause of death (annually 75,000-100,000 deaths in the US) with an economic burden of 184 billion dollars/year in the US. Defining the target(s) and elucidating the molecular mechanism of its action is needed for effective intervention. The objective of this proposal is to define the molecular mechanisms by which alcohols exert their action on intracellular signal transduction pathways in brain. Alcohols are known to alter the expression and activity of Protein Kinase Cs (PKC), a family of kinases mainly expressed in the brain. While PKC epsilon knock-outs showed significant decrease in alcohol consumption and increase in tolerance to ethanol compared to wild type mice, the PKC gamma knock-outs showed significant increase in alcohol consumption and decrease in tolerance. PKC[unreadable] and PKC3 knock-outs also showed opposite properties in regulating responses to GABAA (3-amino butyric acid) receptors. GABAA is a ligand-gated ion channel in brain and believed to be an important target of alcohol. Structurally, PKC3 has its regulatory C1 domain (combination of C1A and C1B) at the N terminus followed by the regulatory C2 domain. On the other hand, PKC[unreadable] has its C2 domain at the N terminus followed by the C1 domain. The C1A and C1B subdomains of epsilon and gamma differ significantly in terms of their ligand binding properties. The central hypothesis to be tested is that the structural and ligand binding differences in the regulatory domains of PKC[unreadable] and PKC3 are responsible for their differential behavioral response to alcohol. The first aim of this proposal is to determine how alcohols regulate PKC[unreadable] and PKC3 activity and if there is an alcohol binding site in their regulatory domains. In order to identify alcohol binding site we will use novel photoactive diazirine analog of alcohols to photolabel the PKC domain/subdomains followed by identification of the labeled residues by mass spectrometry. The second aim is to generate PKC chimeras by swapping the regulatory domains/subdomains between PKC[unreadable] and PKC3 and characterize their alcohol binding properties. This will be achieved by measuring the effect of alcohols on the activities of the chimeric proteins. The third aim is to investigate the effect of alcohols on GABAA-PKC interactions by measuring GABAA current electrophysiologically. The proposal will enhance our current understanding on the molecular mechanism of alcohol action and will provide strategy for designing new therapeutics against alcohol addiction. PUBLIC HEALTH RELEVANCE: Despite huge number of alcohol related deaths (annually 75000-100,000 in the US) and cost to society ($184 billion/yr in the US) very few medications are available for treating alcohol related diseases. To develop new medications and effective prevention it is necessary to define the target and mechanism of its action at the molecular level. The significance of the present study is to establish the role of a signal transducing protein in regulating alcohol actions. This study will enhance the knowledge in developing medication for alcohol addiction.